ORIGINAL ARTICLE

Association of Rsa polymorphism of the estrogen receptor-b genewith rheumatoid arthritis

Hiromi Sato • Ayano Ito • Aranzazu Gonzalez-Canga •

Hiroko Okuzawa • Kanako Ugai • Masahiko Suzuki •

Takao Namiki • Koichi Ueno

Received: 26 November 2010 / Accepted: 13 April 2011 / Published online: 27 April 2011

� Springer-Verlag 2011

Abstract To investigate the possible influence of the

single nucleotide polymorphism (SNP) of the estrogen

receptor-b gene, rs1256049 (Rsa) in exon 5, on the frequency

of rheumatoid arthritis (RA), 263 RA patients and 174 con-

trol subjects with osteoarthritis (OA) were recruited. Rsa

polymorphism was detected using a PCR–RFLP, Polymer-

ase Chain Reaction—Restriction Fragment Length Poly-

morphism method. The occurrence of both mutant allele

(G) and genotype (GG) were significantly higher in RA than

in OA patients (allele P = 0.008, OR: 1.501, 95%CI:

1.12–2.02). In RA patients, GG genotype frequency was

higher in severe RA patients than mild RA patients. More-

over, there was significant difference between severe RA

patients and OA patients (P = 0.009), also the allele distri-

bution was significant different between severe RA, mild

RA, and OA patients (P = 0.025, 95%CI = 0.61–0.93).

With respect to gender, GG genotype was statistically more

frequent in female RA patients than that of OA, while such an

association was not observed in men. Above all, the presence

of the GG genotype could be a risk factor for RA and such

trend might be different in gender, although additional larger

scale study is needed.

Keywords Polymorphism � Rheumatoid arthritis �Sex � Estrogen receptor-b

Introduction

Rheumatoid arthritis (RA) is the most common chronic

autoimmune disorder being present in approximately 1% of

the population worldwide [1, 2]. RA is considered a clinically

heterogeneous condition with a wide spectrum of clinical

manifestations, great variability in severity and disease pro-

gression, and different responses to a range of therapies [1].

Rheumatoid arthritis etiology is complex, in common

with other autoimmune disorders, and implies the interac-

tion between environmental and genetic factors [1].

Regarding the genetic contribution, comparisons of con-

cordance rates in monozygotic and dizygotic twins strongly

support a heritable contribution to susceptibility in RA [3].

The prediction of risk for disease progression is impre-

cise, and is based on a combination of demographic, clinical,

and laboratory factors [4–6]. Epidemiological studies show

an important genetic influence in RA, and in this sense,

genetic tests could be of great value for an early diagnosis.

Regarding gender, RA is affecting approximately twice

as many women as men [1, 2]. Some studies suggest that

female sex hormones and pregnancy are factors possibly

associated with RA symptoms. Thus, it was reported that

amelioration of RA occurs in about three quarters of

pregnancies and most women who improve experience

initial relief in the first trimester, though almost invariably

RA recurs within 3–4 months of delivery [7]. Another

investigation concluded that the menopausal state could be

H. Sato (&) � A. Ito � H. Okuzawa � K. Ugai � K. Ueno

Department of Geriatric Pharmacology and Therapeutics,

Graduate School of Pharmaceutical Sciences, Chiba University,

1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan

e-mail: hiromi-s@p.chiba-u.ac.jp

A. Gonzalez-Canga

Department of Biomedical Sciences, Veterinary Faculty,

University of Leon, Leon, Spain

M. Suzuki

Department of Orthopedic Surgery, Graduate School

of Medicine, Chiba University, Chiba, Japan

T. Namiki

Department of Frontier Japanese-Oriental (Kampo) Medicine,

Graduate School of Medicine, Chiba University, Chiba, Japan

123

Rheumatol Int (2012) 32:2143–2148

DOI 10.1007/s00296-011-1947-2

responsible for the major part of the differences in outcome of

RA between men and women [8]. Furthermore, women RA

patients are likely to have a more severe disease than men [9].

On the other hand, the influence of exogenous hormones have

also been studied. Thus, Hall et al. [10] assessed the effects of

hormone replacement therapy on disease activity in post-

menopausal RA patients and found that estrogens could pos-

sibly suppress inflammatory arthritis. There are also some

studies confirming that women who take the oral contracep-

tive pill are at reduced risk of developing RA, although the

exact mechanisms remain unclear [11, 12].

On the other hand, estrogens could be associated to RA

development by means of regulating bone metabolism and

the immune function [13].

To understand, in detail, the functional role of estrogens

in RA development it is also important not only to study

estrogens, but the estrogen receptor (ER) as well. In the

past, two types of estrogen receptors have been identified

and cloned: estrogen receptor-a (ERa) and estrogen

receptor-b (ERb) [14, 15].

Several studies have verified the association between

polymorphism of the ERb gene (gene located on human

chromosome 14q22-24) [15], such as cytosine-adenine

(CA) dinucleotide repeat, and various diseases, such as

Alzheimer’s disease [16], osteoporosis [17], bulimia [18],

endometriosis [19], and ovulatory dysfunctions [20]. We

have previously reported that longer CA repeat might be a

risk factor for RA [21].

There is also another interesting polymorphism, rs1256049.

This single nucleotide polymorphism (Guanine to Adenine)

exists in exon 5 of ERb gene, and is called Rsa polymorphism

because it is detected using the restriction enzyme Rsa I. Some

reports linked this polymorphism with inflammatory diseases

[22, 23], but direct relationship between Rsa polymorphism

and RA has not yet been established.

In this study, we investigated the association between

Rsa polymorphism in exon 5 of the ERb gene and RA in

both adult men and women, and compared the results

obtained with those found in OA patients.

Materials and methods

Protocols and procedures for this experiment were approved

by the Ethics Committee of the Graduate School of Phar-

maceutical Sciences of Chiba University. All the genetic

information employed in this study remains confidential.

Subjects

A total number of 437 Japanese patients (72 men and 365

women) were recruited to participate in this study from

Chiba University Hospital, Japan.

These patients belong to two groups of subjects: the first

group (n = 263; 53 men and 210 women) was diagnosed

with rheumatoid arthritis, while the second one (n = 174;

19 men and 155 women), that served as control, was

recruited from the same geographical area and suffered

from osteoarthritis. Informed consent was obtained from all

the subjects who participated in this study.

DNA extraction and PCR–RFLP method

Genomic DNA was extracted from peripheral blood leu-

kocytes with a QIAamp DNA Blood Mini Kit (QIAGEN,

Hilden, Germany) according to a standard protocol. Poly-

merase chain reaction (PCR) was performed with oligo-

nucleotide primers designed to amplify a polymorphic site

(Rsa) in exon 5 of the human ERb gene. PCR was per-

formed in a total volume of 25 lL reaction mixture with

the following components: 100 ng of human genomic

DNA, each primer (forward: 50-GTA AAA CGA CGG

CCA GTT CTC ACC GCC TCT TGC TTT CCC CAG-30

and reverse: 50- AAG GGA ACA AAA GCT GGA GAA

ACA CAA TGT ATT TTT TCT CAC-30) at 0.2 lM, and

12 lL of Premix Ex TaqTM Hot Start Version, completing

to 25 lL with distilled water. The amplification was per-

formed at 95�C for 10 min, following 35 cycles: 95�C for

30 s, 58�C for 30 s, and finally 72�C for 10 min. PCR

products were treated with 5 U of Rsa I enzyme at 37�C for

5 h. Finally, the products were electrophoresed through a

3% agarose gel. A DNA product with only 223 bp band

was determined to be G allele (major allele), while a

product with 150 and 73 bp bands was determined as A

allele (minor allele). Representative bands are shown in

Fig. 1. We have previously confirmed that the results of

PCR–FRLP method were consisted with those of DNA

direct sequence method.

Fig. 1 The electrophoretic profiles of rs1256049 (Rsa polymorphism)

by PCR–RFLP. Identification of ‘‘A’’ by Rsa I. The panel shows the

amplified PCR fragments that were digested with restriction enzyme

(?) or (-). M DNA size marker (100 bp DNA ladder), A major

allele ? major allele (GG genotype), B major allele ? minor allele

(GA genotype), C minor allele ? minor allele (AA genotype)

2144 Rheumatol Int (2012) 32:2143–2148

123

Statistical analysis

Regarding to allele or genotype, groups were compared

using the Fisher’s exact probability test. On the other hand,

two groups were compared with the Student’s t-test or, in

case of unequal variance, with the Aspin–Welch t-test

about old comparison. P-values B 0.05 was considered to

represent a statistically significant for all the analyses.

Results

The patients profile is shown in Table 1. Significant dif-

ferences were detected between RA and OA in gender,

although both in RA and OA female ratio was higher than

that of men. RA patients were younger than OA patients

(RA: women 60.4 ± 11.6 years, men 60.6 ± 12.2 years;

OA: women 68.2 ± 9.2 years, men 68.5 ± 13.6 years).

Finally, dealing with the severity degree of RA, we used

the same criteria as Ochi et al. [24] to classify our patients

into three types (the subset with least erosive disease LES;

the subset with more erosive disease: MES; and the subset

with mutilating disease: MUD), as it is shown in Table 1.

These types are determined by orthopedic specialists from

long-time observation. The important point is how the joint

destructions progress; mildly or rapidly, oligoarthritis or

multi joint, so it does not depend on the sensitivity to

treatment drugs. This is the reason why we selected this

classification for retrospective study. As a result, most of

the patients were medium type, MES.

The frequency distribution of Rsa genotype and alleles

in RA and OA patients were significantly different as can

be observed in Table 2 (Allele P = 0.008, OR = 1.501,

95% CI = 1.12–2.02).

Though GG genotype was the more common in both RA

and OA, the frequency was significantly higher in RA

compared to OA (P = 0.008, OR = 1.685, 95%

CI = 1.15–2.48) (Table 3). The observed genotype fre-

quencies were in Hardy–Weinberg equilibrium.

To further investigate the observed association, our RA

patients were classified and divided into two groups

(according to the severity degree): mild patients (LES) and

severe patients (MES ? MUD). We found that the fre-

quency of GG genotype followed the next order: severe RA

patients, mild RA patients, and OA patients (Table 4). The

frequency of GG genotype was significantly different

between severe RA patients and OA patients (P = 0.009,

by Fisher’s exact probability test and Bonferroni’s cor-

rection). There was also a significant difference if three

groups were compared (P = 0.025, 95% CI = 0.61–0.93)

(Table 4).

With respect to gender, GG genotype was statistically

more frequent in female RA patients than in OA subjects,

while such an association was not determined in men

(Table 5, the top part). On the basis of this result, we

classified female RA patients according to the severity of

the degree and compared them with OA patients. We found

a significant difference between severe RA patients and

OA patients (P = 0.022, 95% CI = 0.59–0.92) (Table 5,

the bottom part).

Table 1 Characteristics of 263 RA patients and 174 OA patients

Characteristic Total (n (%)) RA (n (%)) OA (n (%)) P-value

All patients

437 263 174 –

Gender

Women 365 (83.5) 210 (79.8) 155 (89.1) 0.012a

Men 72 (16.5) 53 (20.2) 19 (10.9)

Age (mean ± SD)

Women (range) 63.7 ± 11.5 (18–94) 60.4 ± 11.6 (18–86) 68.2 ± 9.2 (37–94) \0.001b

Men (range) 62.7 ± 13.0 (23–84) 60.6 ± 12.2 (23–83) 68.5 ± 13.6 (37–84) \0.05c

Total (range) 63.6 ± 11.6 (18–94) 60.5 ± 11.7 (18–86) 68.3 ± 10.2 (34–94) \0.001b

Severity

LES – 69 (26.2) – –

MES – 183 (69.6) –

MUD – 11 (4.2) –

Age data are expressed as mean ± SD

P-value is shown as comparison between RA and OA by a Fisher’s exact probability test, b Aspin–Welch t-test, c Student’s t-test

Differences were considered significant at P B 0.05

LES least erosive subset, MES more erosive subset, MUD mutilating disease

Rheumatol Int (2012) 32:2143–2148 2145

123

Discussion

To our knowledge, this is the first study to investigate the

association between Rsa polymorphism of the ERb gene

and RA. Our small-scale preliminary results showed that

Rsa polymorphism was positively associated with RA (GG

genotype was more frequent in RA patients), so we ana-

lyzed the possible association between Rsa polymorphism

frequency and RA.

Compared to OA subjects, GG genotype was signifi-

cantly more frequent in RA patients. Our data for Rsa

genotype frequency in OA patients was similar to those of

healthy Japanese, as reported in HapMap–JPT data

(genotypes: GG 45.5%; GA 40.9%; AA 13.6%,

P = 0.878), Rsa genotype frequency of our RA patients

was different from that (P = 0.097) [25].

We further classified our RA patients taking into

account the severity degree. We found no relation between

the severity degree and genotype frequency among our RA

patients, but significant differences between the subgroups

of severe RA patients and OA patients in the frequency of

GG genotype. Moreover, GG genotype was statistically

more frequent in female RA patients than that found in OA

patients. Our results suggest that GG genotype of Rsa

polymorphism is, at least in part, implicated in the occur-

rence of severe RA in female subjects.

Though there are no specific reports establishing a direct

relationship between ERb Rsa polymorphism and RA,

Karlson et al. [26] suggested that hormone-related genes

including estrogen receptor 2 (ERb) are not important to a

risk factor of RA in their prospective study. It was a hap-

lotype analysis of selected five SNPs, rs3020450,

rs1256031, rs1256049 (Rsa), G1730A, and rs944459,

which have been identified to capture the genetic variation

in Caucasians, however, these SNPs are not reported fre-

quently in Japanese except for rs1256049 (Rsa) and

rs944459 [27]. There might be a race difference in ERbpolymorphism, so further study is needed to clarify the

meaning of this genotype with Japanese people.

On the other hand, it is generally accepted that estrogens

influence RA pathogenesis or progression. One of the

possible effects of estrogens on RA could be related to

bone turnover. Nevertheless, it was reported that Rsa

polymorphism was probably not an important determinant

of bone mineral density [28], so we speculated that this

polymorphism could be rather related to immunological

mechanisms of RA development.

In another investigation, Rsa polymorphism was sug-

gested to be associated with risk of cardiovascular disease

(CVD) in female patients. Rexrode et al. indicated that

CVD women patients were less likely to have the Rsa

polymorphism variant A allele (P = 0.004); so the Rsa

variant G allele was a risk determinant. These authors also

found that a common haplotype that included Rsa poly-

morphism was associated with a sevenfold increased risk of

myocardial infarction in women [22]. There was also a

similar work dealing with several haplotypes, including

Rsa polymorphism, that showed an increased risk of

endometrial cancer [23].

On the other hand, the Genome-Wide Association

(GWA) study, reported by the Welcome Trust case Control

Consortium (WTCCC), identified some independent

Table 2 Comparison of genotype and allele frequencies for Rsa

polymorphism of ERb gene in RA and OA patients

Genotype RA OA Genotype Allele G vsAllele A

n (%) n (%) P-value OR 95%CI P-value

GG 149 (56.7) 76 (43.7) 0.026 1.501 1.12–2.02 0.008

GA 94 (35.7) 78 (44.8)

AA 20 (7.6) 20 (11.5)

Total (n) 263 174

Differences were considered significant at P B 0.05 by Fisher’s exact test

OR odds ratio, 95% CI 95% confidence interval

Table 3 Comparison of genotype frequencies for Rsa polymorphism

of ERb gene in RA and OA patients between two groups of genotypes

Genotype RA OA OR 95%CI P-value

n % n %

GG 149 (56.7) 76 (43.7) 1.685 1.15–2.48 0.008

GA ? AA 114 (43.3) 98 (56.3)

Total (n) 263 174

GG ? GA 243 (92.4) 154 (88.5)

AA 20 (7.6) 20 (11.5) 1.578 0.82–3.03 0.178

Total (n) 263 174

OR odds ratio, 95% CI: 95% confidence interval

Differences were considered significant at P B 0.05 by Fisher’s exact

test

Table 4 Comparison of genotype frequencies for Rsa polymorphism

of ERb gene between OA patients, mild (LES) RA patients and severe

(MUD?MES) RA patients

Genotype OA RA (LES) RA(MES ? MUD)

95%CI P-value

n (%) n (%) n (%)

GG 76 (43.7) 37 (53.6) 112 (57.7) 0.61–0.93 0.025

GA ? AA 98 (56.3) 32 (46.4) 82 (42.3)

Total (n) 174 69 194

Differences were considered significant at P B 0.05 by Fisher’s exact test

95% CI 95% confidence interval

LES least erosive subset, MES more erosive subset, MUD mutilating disease

2146 Rheumatol Int (2012) 32:2143–2148

123

signals in common human diseases including RA [29]. In

their study, almost all of these signals reflect genuine

susceptibilities, which supported thorough validation of the

GWA approach. Their GWA study detected three RA

involving SNPs. Interestingly, rs11761231, one of the RA

involved SNPs, was reported to have no effect on disease

status in men, but a strong apparently additive effect in

women. Also, they suggested that it might represent one of

the first sex-differentiated effects in human diseases.

Regarding to the GWA study, it is suggested that some

other SNPs around Rsa probably make linkage disequi-

librium block which is associated to RA. Moreover, this

association might be different between men and women.

Another immunological factor related to RA is TNF-a.

The hyperplasia of the synovial membrane imposed by pro-

inflammatory cytokines has been suggested to play a cru-

cial role in the progression of this disease. Among these

cytokines, TNF-a has a potent effect and was detected at

highly enhanced concentration in the blood and synovial

fluids of RA patients relative to OA patients and normal

subjects [30]. On the other hand, estrogens have significant

anti-inflammatory properties and ERb is a more desirable

therapeutic target rather than ERa because it also mediates

the proliferative effects of estrogens on the mammary

gland and uterus. Some selective ERb agonists like ERB-

041 and WAY-202196 are reported to have potent anti-

inflammatory effects in animal models for diseases [31–

34]. Cvoro et al. [32] suggested that these ERb selective

compounds exerted their anti-inflammatory effects par-

tially by causing transcriptional repression of pro-inflam-

matory genes in immune cells. In their study, ERB-041 was

very effective at transcriptional repression of TNF-a in the

presence of ERb but not ERa. Moreover, ERB-041 has

potent anti-inflammatory activity in the Lewis rat model of

adjuvant-induced arthritis [34]. But, a recent report dem-

onstrated that a specific ERa agonist, PPT dramatically

decreased the frequency and severity of arthritis in mouse

model of collagen-induced arthritis although DPN; for ERbagonist, had no effect [35]. Engdahl et al. [35] suggested

some explanations about these different results, since the

two studies performed in different animal species (rat or

mouse), models (adjuvant or collagen induction), sexes

(male rat or female rat), different ERb agonists or routes,

and so on. It still be unknown which ER molecule is mainly

responsible for RA, however, there is a possibility that Rsa

polymorphism changes ERb function, which might affect

TNF-a transcriptional activity resulting in some effect on

RA developing. Such an investigation would be beneficial

for ER targeting drug discovery.

In our study, Rsa genotype was significantly different from

that of healthy Japanese people and GG genotype was more

frequent in RA patients than in the control group, OA patients.

Although additional larger scale study and further investiga-

tions regarding the involvement to another SNPs are needed,

this polymorphism would be an important prediction marker

in RA development and could be related to sex differences.

Table 5 Comparison of genotype frequencies for Rsa polymorphism of ERb gene between women and men

Genotype RA OA OR 95%CI P-value

n % n %

Women GG 115 (54.8) 65 (41.9) 1.676 1.06–2.01 0.020

GA ? AA 95 (45.2) 90 (58.1)

Total (n) 210 155

Men GG 34 (64.2) 11 (57.9)

GA ? AA 19 (35.8) 8 (42.1) 1.301 0.45–3.79 0.783

Total (n) 53 19

Genotype OA RA (LES) RA (MES ? MUD) 95%CI P-value

n % n % n %

Women GG 65 (41.9) 25 (47.2) 90 (57.3) 0.59–0.92 0.022

GA ? AA 90 (58.1) 28 (52.8) 67 (42.7)

Total (n) 155 53 157

Men GG 11 (57.9) 12 (75.0) 22 (59.5) 0.58–1.80 0.561

GA ? AA 8 (42.1) 4 (25.0) 15 (40.5)

Total (n) 19 16 37

Differences were considered significant at P B 0.05 by Fisher’s exact test

OR odds ratio, 95%CI: 95% confidence interval

LES least erosive subset, MES more erosive subset, MUD Mutilating disease

Rheumatol Int (2012) 32:2143–2148 2147

123

Acknowledgments We are truly thankful to Dr. Yoichi Suzuki in

Medical Faculty of Chiba University for special help of our genotype

analysis. Grant-in-Aid for Scientific Research (C) from the Japan

Society for the Promotion of Sciences and Health Labor Sciences

Research Grant.

Conflict of interest The authors declare that they have no conflict

of interest.

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